2.1 | Design and synthesis of functionalized PEG-lipids
We designed and prepared PCNDs conjugated with cargo proteins and 9E5 using end-functionalized poly(ethylene glycol)(PEG)-lipids (Figure 1A and 1B). The PCNDs used in this study consisted of an outer lipid coating and an inner perfluorocarbon core. As previously reported[30,31], dipalmitoyl phosphatidylcholine (DPPC) and its PEGylated analog (DPPE-PEG-OMe) were used as the basic lipid components (Figure 1B). Perfluorohexane (PFH) was employed as the perfluorocarbon based on a pioneering report on PCND-mediated endosomal rupture[28]. The lifetime of microbubbles generated from PCNDs with the PFH core was on the order of microseconds, and after ultrasound-induced vaporization, the bubbles were reported to disappear within 10 μs by re-condensation[32]. Accordingly, in the reported endosomal rupture system, PCNDs were assumed to vaporize only for a moment inside the cells, thereby avoiding significant cell damage (Figure 1C)[28]. In our previous studies[30,31], biotinylated antibody 9E5 was conjugated with biotinylated PEG-lipid (DPPE-PEG-biotin) by crosslinking with a streptavidin analog, NeutrAvidin, on the lipid coating of PCNDs (Figure 1B). Additionally, in this study, cargo proteins were designed to conjugate onto the PCND surface via a disulfide linker because they were required to be released in the reductive intracellular environment. Therefore, a PEG-lipid functionalized with 3-[(2-pyridyl) dithio] propionyl (DPPE-PEG-PyDTP) moiety was synthesized (see Supporting Information, Figure S1 and S2) and incorporated into the lipid coating of the PCNDs (Figure 1A). In the present design, the thiol group of the proteins reacted with DPPE-PEG-PyDTP, resulting in conjugation via thiol-disulfide exchange (Figure 1A).